Polysaccharide-protein conjugate vaccines

ABSTRACT

Methods for synthesis and manufacture of polysaccharide-protein conjugate vaccines at high yield are provided. The methods involve reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant. The reaction proceeds rapidly with a high conjugation efficiency, such that a simplified purification process can be employed to separate the conjugate product from the unconjugated protein and polysaccharide and other small molecule by-products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. patent application Ser. No. 13/892,163,filed May 10, 2013, which is a Divisional of U.S. patent applicationSer. No. 13/243,480, filed Sep. 23, 2011, issued as U.S. Pat. No.8,465,749, which is a Divisional of U.S. patent application Ser. No.10/566,899, filed Sep. 25, 2006, issued as U.S. Pat. No. 8,048,432,which is the U.S. National Stage of International Application No.PCT/US04/25477, filed on Aug. 6, 2004, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Patent Application No. 60/493,389, filed Aug. 6, 2003, allof which are hereby incorporated by reference.

FIELD OF THE INVENTION

Methods for synthesis and manufacture of polysaccharide-proteinconjugate vaccines at high yield are provided. The methods involvereaction of a hydrazide group on one reactant with an aldehyde orcyanate ester group on the other reactant. The reaction proceeds rapidlywith a high conjugation efficiency. Simplified purification processescan be employed to separate the conjugate product from the unconjugatedprotein and polysaccharide and other small molecule by-products.

BACKGROUND OF THE INVENTION

Bacterial polysaccharides (PSs) are T-independent antigens inducingshort-term immunity in older children and adults, but frequently not inyoung infants. PSs are incapable of binding to the majorhistocompatibility complex molecules, which is required for antigenpresentation to and stimulation of T-helper lymphocytes. PSs are able tostimulate B lymphocytes for antibody production without the help ofT-helper lymphocytes. As a result of the T-independent stimulation ofthe B lymphocytes, there is a lack of memory induction followingimmunization by these antigens.

T-independent polysaccharide antigens can be converted to T-dependentantigens by covalent attachment of the polysaccharides to proteinmolecules. B cells that bind the polysaccharide component of theconjugate vaccine can be activated by helper T cells specific forpeptides that are a part of the conjugated carrier protein. The T-helperresponse to the carrier protein serves to augment the antibodyproduction to the polysaccharide. PS-conjugate vaccines arepolysaccharide-protein hybrids formed by the covalent attachment of aprotein to a PS. Chemical modification of the PS prior to attachment istypically required because most native bacterial PSs cannot bechemically linked to a protein without first undergoing some chemicalmodification (“activation”).

Attachment to the protein yields a number of T cell epitopes. These Tcell epitopes interact with CD4 helper T cells, greatly facilitating anantibody response to the attached polysaccharide. The T helpercell-dependent response to a conjugate results in both serum IgGantibodies and immune memory, even in infants. Additionally, theimmunogenicity of the PS-conjugate, in contrast to the native PS, isless dependent on the size of the conjugated PS. Accordingly, conjugatesprepared with either PS or oligosaccharides can have similarimmunogenicity.

There are many conjugation reactions that have been employed forcovalently linking polysaccharides to proteins. Three of the morecommonly employed methods include: 1) reductive amination, wherein thealdehyde or ketone group on one component of the reaction reacts withthe amino or hydrazide group on the other component, and the C═N doublebond formed is subsequently reduced to C—N single bond by a reducingagent; 2) cyanylation conjugation, wherein the polysaccharide isactivated either by cyanogens bromide (CNBr) or by1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate (CDAP) tointroduce a cyanate group to the hydroxyl group, which forms a covalentbond to the amino or hydrazide group upon addition of the proteincomponent; and 3) a carbodiimide reaction, wherein carbodiimideactivates the carboxyl group on one component of the conjugationreaction, and the activated carbonyl group reacts with the amino orhydrazide group on the other component. These reactions are alsofrequently employed to activate the components of the conjugate prior tothe conjugation reaction.

The Haemophilus influenzae type b (Hib) conjugate vaccines represent thefirst PS-protein conjugate vaccines produced for clinical use. Robbinsand his colleagues in 1980 utilized the biotechnological process ofchemically attaching saccharides to protein carriers, a conceptdeveloped 50 years earlier. See Avery et al., J. Exp. Med. 1929;50:533-550; Schneerson et al., J. Exp. Med 1980; 152:361-376. There arenow four different Hib conjugate vaccines licensed in the United States,each different, and each having their own physical, chemical, andimmunological characteristics, as summarized in Table 1. A detailedreview of the conjugation chemistry and quality control used in thesevaccines has been published. See Kniskern et al., “Conjugation: design,chemistry, and analysis” in Ellis et al., Development and clinical usesof Haemophilus b conjugate vaccines. New York: Marcel Dekker, 1994:37-69.

TABLE 1 Vaccine* Saccharide size Carrier protein Spacer (linker) PRP-DPolysaccharide Diphtheria toxoid 6-carbon spacer (Connaught) (ADH) HbOCOligosaccharide Diphtheria protein None (amide) (Wyeth-Lederle) (CRM)PRP-OMPC Small Meningococcal Thioether (Merck) polysaccharide protein(bigeneric) PRP-T polysaccharide Tetanus toxoid 6-carbon spacer (AventisPasteur) (ADH) *The four Hib conjugate vaccines are described commonlyin the literature with these acronyms and the responsible manufacturersare in parentheses.

The first commercial Hib conjugate, polyribosylribitol phosphatediphtheria toxoid conjugate (PRP-D), consists of partially size-reducedHib PS attached through a six-carbon spacer, adipic acid dihydrazide(ADH), to diphtheria toxoid using the procedure of Schneerson et al., J.Exp. Med. 1980; 152:361-376. The ADH derivative of diphtheria toxoid wasobtained in this method by reaction with ADH in the presence of1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (EDC).The Hib PS was then activated by creating cyanate groups on the hydroxylgroups using CNBr. The activated PS was conjugated to the ADH-toxoid(cyanylation conjugation), but the process created an unstable linkageand the conjugate had solubility problems.

The Robbins conjugation chemistry was later modified such that the ADHspacer is added first to the polysaccharide, which is then conjugated tothe purified protein in the presence of EDC (carbodiimide reaction). SeeChu et al., Infect. Immun. 1983; 40:245-256; Schneerson et al. Infect.Immun. 1986, 52:519-528. This modification improved the conjugationefficiency and product solubility. The vaccine polyribosylribitolphosphate tetanus protein conjugate (PRP-T) utilizes the improvedchemistry to covalently link Hib polysaccharide to tetanus toxoid (seeTable 1).

The polyribosylribitol phosphate cross reacting mutant diphtheria toxoidconjugate (PRP-CRM) vaccine, also referred to as Haemophilus boligosaccharide conjugate (HbOC), does not contain Hib PS. Instead, itutilizes oligosaccharides of about repeat units derived by periodateoxidation of the glycol functionality in the ribitol moiety. Theoxidized oligosaccharides are then attached directly to CRM₁₉₇ anontoxic mutant form of diphtheria toxin isolated from cultures ofCorynebacterium diphtheriae C7 (f3197), in the presence of sodiumcyanoborohydride (reductive amination). See Anderson et al., J. Immunol.1989; 142:2464-8; and Anderson, Infect. Immun. 1983, 39:233-238. In thisconjugation method, the ratio of oligosaccharide to protein was found tobe critical for optimal antibody response. See Kniskern et al.,“Conjugation: design, chemistry, and analysis” in Ellis et al.,Development and clinical uses of Haemophilus b conjugate vaccines. NewYork: Marcel Dekker, 1994: 37-69; Anderson et al., J. Immunol. 1989;142:2464-8.

Compared to the other Hib conjugate vaccines, Hibpolysaccharide-Neisseria meningitidis outer membrane protein complexconjugate vaccine (PRP-OMPC) has a number of unique properties. Theprotein carrier is not a component of the diphtheria, tetanus, andpertussis (DTP) vaccine, but consists of lipopolysaccharide-depletedmeningococcal outer membrane vesicles to which are attached size-reducedHib PS through a thioether linkage. See Marburg et al., J. Amer. Chem.Soc. 1986; 108:5282-5287; Kniskern et al., “Conjugation: design,chemistry, and analysis” in Ellis et al., Development and clinical usesof Haemophilus b conjugate vaccines. New York: Marcel Dekker, 1994:37-69; Anderson et al., J. Immunol. 1989; 142:2464-8. In this process,separate linkers are attached to both the protein and Hibpolysaccharide, followed by fusion of the linkers to form a thioetherlinkage.

Neisseria meningitidis is a leading cause of bacterial meningitis andsepsis throughout the world. Pathogenic meningococci are enveloped by apolysaccharide capsule that is attached to the outer membrane surface ofthe organism. Thirteen different serogroups of meningococci have beenidentified on the basis of the immunological specificity of the capsularpolysaccharide (Frasch, C. E., et. al., “Serotype antigents of Neisseriameningitides and a proposed scheme for designation of serotypes,” RevInfect Dis. 7(4):504-10, July-August 1985). Of these thirteenserogroups, five cause the majority of meningococcal disease; theseinclude serogroups A, B, C, W135, and Y. Serogroup A is responsible formost epidemic disease. Serogroups B, C, and Y cause the majority ofendemic disease and localized outbreaks. Host defense of invasivemeningococci is dependent upon complement-mediated bacteriolysis. Theserum antibodies that are responsible for complement-mediatedbacteriolysis are directed in large part against the outer capsularpolysaccharide.

Conventional vaccines based on meningococcal polysaccharide elicit animmune response against the capsular polysaccharide. These antibodiesare capable of complement-mediated bacteriolysis of the serogroupspecific meningococci. The meningococcal polysaccharide vaccines wereshown to be efficacious in children and adults. However, efficacy waslimited in infants and young children, and subsequent doses of thepolysaccharide in younger populations elicited a weak or no boosterresponse.

There are a number of approaches that have been employed for activationof the meningococcal PS and for conjugation, as summarized in Table 2.Each mode of activation has the potential to alter important epitopes,even when relatively few sites are activated on the PS molecule.Periodate activation of the group C meningococcal PS, for example,results in chain breakage generating smaller saccharide units withterminal aldehyde groups that can be linked to the protein via reductiveamination. Richmond et al., J. Infect. Dis. 1999; 179:1569-72.

TABLE 2 Carrier Used in Method Saccharide size protein Spacer Procedurehumans #1 Reduced Tetanus None Aldehyde form of PS No Reductive toxoidcombined with protein amination in presence of sodium cyanoborohydride#2 Native Tetanus None PS and protein No Carbodiimide toxoid combined inpresence of carbodiimide, then blocked with ethanolamine #3Oligosaccharide CRM₁₉₇ Adipic Aminated reducing Yes Active ester^(a)acid terminus of the oligosaccharide conjugated to protein by adipicacid (NHS)₂ #4 Reduced CRM₁₉₇ None Aldehyde form of Yes Reductivesaccharide combined amination with protein in presence of sodiumcyanoborohydride #5 De-OAc PS^(b) Tetanus None Aldehyde form of PS YesReductive toxoid combined with protein amination in presence of sodiumcyanoborohydride ^(a)N-hydroxysuccinimide diester of adipic acid^(b)Deacetylylated PS only reported for Meningococcal group C

Initial studies on production and optimization of meningococcal group Cconjugates were reported well before commercialization of the Hibconjugates. See Beuvery et al., Infect. Immun. 1982; 37:15-22; Beuveryet al., Infect. Immun. 1983; 40:39-45; Beuvery et al., J. Infect. 1983;6:247-55; Jennings, et al., J. Immunol. 1981; 127:1011-8.

Two different conjugation methodologies have been reported forchemically linking the group C PS to a protein carrier. See Jennings etal., J. Immunol. 1981; 127:1011-8; Beuvery et al., Infect. Immun. 1983;40:39-45. The first approach employs partially depolymerized PS, whichis activated by creation of terminal aldehyde groups through periodateoxidation (Method #1 in Table 2). The aldehydes are then reacted throughreductive amination combined with free amino groups on the protein,mostly lysines, in the presence of sodium cyanoborohydride. See Jenningset al., J Immunol 1981; 127:1011-8. In this method, activation occurs atone specific site on the group C PS.

The second approach utilizes the carbodiimide reaction (Method #2 inTable 2) to covalently link carboxylic groups in the high molecularweight PS to lysine ε-amino groups on the carrier protein. Theactivation sites in this method are more random, compared to periodateactivation.

Group C meningococcal conjugates prepared by these two methods have beenevaluated in animals. See Beuvery et al., Dev. Biol. Stand. 1986;65:197-204; and Beuvery et al., J. Infect. 1983; 6:247-55. Theconjugates stimulated both T cell independent and T cell dependentresponses upon initial immunization. See Beuvery et al., J. Infect.1983; 6:247-55. Studies have shown that the PS must, however, becovalently linked to the carrier protein to induce a T cell dependentantibody response.

The first group A and group C meningococcal conjugates to be used inclinical trials were prepared by Chiron Vaccines and were reported in1992 (Method #3 in Table 2). See Costantino et al., Vaccine 1992;10:691-8. The conjugation method was based upon selective terminal groupactivation of small oligosaccharides produced by mild acid hydrolysisfollowed by coupling to a protein through a hydrocarbon spacer. Thenon-toxic mutant of diphtheria toxin, CRM₁₉₇, was used as the proteincarrier. To activate the oligosaccharides for conjugation, an aminogroup was added to the end of the oligosaccharide, and then reacted withthe N-hydroxysuccinimide diester of adipic acid to create an activeester. This active ester was then covalently bound to lysine s-aminogroups in the CRM₁₉₇ protein, creating the conjugate.

SUMMARY OF THE INVENTION

Conventional methods for the preparation of PS-protein conjugatevaccines do not use hydrazide chemistry in the reductive aminationconjugation reaction, even though hydrazide in the form of ADH has beenused in activating polysaccharide. These prior art methods utilizeε-amino groups of lysine residues on the protein to react withfunctional groups on activated PSs, such as aldehyde groups (reductiveamination) and carboxyl groups. The efficiency of the reaction is low,typically only about 20%. The reaction also requires two to three daysfor the conjugation to be completed, necessitating the use ofpurification steps to separate the conjugate from unreacted PS. See Guoet al., “Protein-polysaccharide conjugation” in: Pollard et al., Methodsin Molecular Medicine, Vol. 66: Meningococcal Vaccines: methods andProtocols, Humana Press, Totowa, N.J., 2001, pg 49-54. There are anumber of explanations that have been proposed for the low yieldsobserved. First, the ε-amino group of lysine (pKa=10.5) has lowreactivity at the conjugation conditions (pH 6.5-7.4). See Inman et al.,Biochemistry 1969; 8:4074-4082. Secondly, most conjugation methodsemploy toxoids as the carrier proteins. The toxoids are derived from atoxin by detoxification with formaldehyde, which combines with the aminogroups of the toxin, leaving a limited numbers of amino groups availablefor conjugation. Thirdly, reduced solubility of the resulting activatedprotein and protein-PS conjugate can lead to precipitation.

Accordingly, methods for the synthesis and manufacture ofpolysaccharide-protein conjugate vaccines in high yields are desirable.Also desirable are methods wherein the reaction proceeds at a rapidrate, with reduced production of undesired by-products, and with reducedamounts of unreacted protein and polysaccharide remaining at the end ofthe reaction.

Existing vaccines based on PSs are of limited use in young children anddo not provide long-lasting protection in adults. Thus, a need existsfor a protein-PS conjugate vaccine capable of conferring long termprotection against diseases in children and adults at risk for, e.g.,bacterial meningitis, influenza, tetanus, and other bacterialinfections. The protein-PS conjugates of the preferred embodiment can beemployed to prepare vaccine formulations capable of conferring long termprotection to infants, children, and adults.

Accordingly, in a first embodiment, a method for preparing a conjugatevaccine is provided, the method comprising reacting a polysaccharidewith an oxidizing agent, whereby a solution of an aldehyde-activatedpolysaccharide is obtained; buffer exchanging the solution of thealdehyde-activated polysaccharide to a pH of from about 7 to about 8;reacting a protein with hydrazine or adipic acid dihydrazide in thepresence of 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimidehydrochloride at a pH of from about 6 to about 7, whereby a solution ofan hydrazide-activated protein is obtained; raising a pH of the solutionof the hydrazide-activated protein to from about 7.0 to about 11; bufferexchanging the solution of the hydrazide-activated protein to a pH offrom about 10.0 to about 11.0; reacting the aldehyde-activatedpolysaccharide with the hydrazide-activated protein at a pH of fromabout 6 to about 8, whereby a conjugate comprising one or more C═Ndouble bonds is obtained; and reducing substantially all of the C═Ndouble bonds of the conjugate to C—N single bonds, whereby a conjugatevaccine capable of stimulating an immune response is obtained.

In an aspect of the first embodiment, the oxidizing agent comprisesNaIO₄.

In an aspect of the first embodiment, the solution of thealdehyde-activated polysaccharide is buffer exchanged with a HEPESbuffer.

In an aspect of the first embodiment, the solution of thehydrazide-activated protein is buffer exchanged with a Na₂CO₃ buffer.

In an aspect of the first embodiment, the aldehyde-activatedpolysaccharide is reacted with the hydrazide-activated protein at aratio of from about 1:2 to about 2:1.

In an aspect of the first embodiment, reducing comprises reducing withNaBH₄.

In an aspect of the first embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the first embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

In a second embodiment, a method for preparing a conjugate vaccine isprovided, the method comprising reacting a polysaccharide with1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate, whereby asolution of a cyanate-activated polysaccharide is obtained; reacting aprotein with hydrazine or adipic acid dihydrazide in the presence of1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride at a pHof from about 6 to about 7, whereby a solution of a hydrazide-activatedprotein is obtained; raising the pH of the solution of thehydrazide-activated protein to from about 7.0 to about 11; bufferexchanging the solution of the hydrazide-activated protein to a pH offrom about 10.0 to about 11.0; reacting the cyanate-activatedpolysaccharide with the hydrazide-activated protein at a pH of fromabout 6 to about 8 to yield a conjugate vaccine capable of stimulatingan immune response.

In an aspect of the second embodiment, the step of reacting thecyanate-activated polysaccharide with the hydrazide-activated protein isconducted in the absence of a blocking agent.

In an aspect of the second embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the second embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

In a third embodiment, a method for preparing a conjugate vaccine isprovided, the method comprising reacting a protein with1-amino-2,3-propanediol (APDO) in the presence of1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride at a pHof from about 6 to about 7, whereby a solution of a APDO-modifiedprotein is obtained; buffer exchanging the solution of the APDO-modifiedprotein to a pH of from about 10.0 to about 11.0; reacting theAPDO-modified protein with an oxidizing agent, whereby a solution of analdehyde-activated protein is obtained; buffer exchanging the solutionof the aldehyde-activated protein to a pH of from about 10.0 to about11.0; reacting a hydrazide-activated polysaccharide with thealdehyde-activated protein at a pH of from about 6 to about 8, whereby aconjugate comprising one or more C═N double bonds is obtained; andreducing substantially all of the C═N double bonds of the conjugate toC—N single bonds, whereby a conjugate vaccine capable of stimulating animmune response is obtained.

In an aspect of the third embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the third embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

In an aspect of the third embodiment, the hydrazide-activatedpolysaccharide is prepared by reacting a polysaccharide with anoxidizing agent in a solution, whereby an aldehyde-activatedpolysaccharide is obtained; reacting the aldehyde-activatedpolysaccharide with adipic acid dihydrazide to yield an intermediatecomprising one or more C═N double bonds; and reducing substantially allof the C═N double bonds of the intermediate to C—N single bonds, wherebya hydrazide-activated polysaccharide is obtained.

In an aspect of the third embodiment, the hydrazide-activatedpolysaccharide is prepared by reacting a polysaccharide with1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate, whereby acyanate-functionalized polysaccharide is obtained; reacting thecyanate-functionalized polysaccharide with adipic acid dihydrazide,whereby a hydrazide-activated polysaccharide is obtained.

In an aspect of the third embodiment, the hydrazide-activatedpolysaccharide is prepared by reacting a polysaccharide with adipic aciddihydrazide in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, whereby a hydrazide-activated polysaccharideis obtained.

In a fourth embodiment, a conjugate vaccine is provided, the conjugatevaccine comprising at least one polysaccharide moiety and at least oneprotein moiety, wherein the polysaccharide moiety is linked to theprotein moiety through at least one linking group of the formula—C(═O)—NH—NH—CH₂—.

In an aspect of the fourth embodiment, the conjugate vaccine comprises aplurality of polysaccharide moieties and a plurality of protein moietiescrosslinked to form a lattice structure by a plurality of linkinggroups.

In an aspect of the fourth embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the fourth embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

In a fifth embodiment, a conjugate vaccine is provided, the conjugatevaccine comprising at least one polysaccharide moiety and at least oneprotein moiety, wherein the polysaccharide moiety is linked to theprotein moiety through at least one linking group of the formula—C(═O)—NH—NH—C(═NH)—O—.

In an aspect of the fifth embodiment, the conjugate vaccine comprises aplurality of polysaccharide moieties and a plurality of protein moietiescrosslinked to form a lattice structure by a plurality of linkinggroups.

In an aspect of the fifth embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the fifth embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

In a sixth embodiment, a conjugate vaccine is provided, the conjugatevaccine comprising at least one polysaccharide moiety and at least oneprotein moiety, wherein the polysaccharide moiety is linked to theprotein moiety through at least one linking group of the formula—C(═O)—NH—CH₂—CH₂—NH—NH—.

In an aspect of the sixth embodiment, the conjugate vaccine comprises aplurality of polysaccharide moieties and a plurality of protein moietiescrosslinked to form a lattice structure by a plurality of linkinggroups.

In an aspect of the sixth embodiment, the polysaccharide is selectedfrom the group consisting of Meningococcal polysaccharides, Pneumococcuspolysaccharides, Hemophilus influenzae type b polysaccharide, Vipolysaccharide of Salmonnella typhi, and group B Streptococcuspolysaccharides.

In an aspect of the sixth embodiment, the protein is selected from thegroup consisting of tetanus toxoid, diptheria toxoid, CRM₁₉₇, andmeningococcal protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides spectra of products of the conjugation of periodateactivated group C meningococcal polysaccharide to a) ε-amino groups onlysines (TT) (conventional method), and b) hydrazide groups on asparticand glutamic acid residues (TT-H). The spectra are taken fromconjugation products before a dialysis step and contain extra peaks atgreater than 25 minutes not seen after dialysis. The yield of theconjugate (Conj.) is much greater for TTH than TT.

FIG. 2 provides high-performance size exclusion chromatography (HPSEC)profiles of Pn 18C PS-TT conjugates prepared by cyanylation conjugationin the absence and presence of a blocking agent ADH, hydrazine, glycineor ethanolamine. The conjugate peak (Conj., 15.5 minutes) is reducedsignificantly in the presence of a blocking agent while the free proteinpeak (22 minutes) is not. The spectra are taken from conjugationproducts before the dialysis step and contain extra peaks at greaterthan 25 minutes not seen after dialysis.

FIG. 3 provides HPSEC profiles of four Mn C PS-TT conjugates prepared byreductive amination conjugation of aldehyde-activated PS andhydrazide-activated protein. The HPSEC profiles shift slightly atdifferent time. The right shoulder at 22.5-24 minutes is from theunconjugated protein TTH or TTADH, while the left shoulder at 16-17minutes is from high molecular weight conjugate.

FIG. 4 provides estimation of free polysaccharide in a Mn C PS-TTconjugate product prepared by reductive amination conjugation ofaldehyde-activated PS and hydrazide-activated protein. FIG. 4A providesHPSEC profiles of an Mn C PS-TT conjugate pre (3) and post (1) C18absorption, and pure TTH (2) monitored at 280 nm, detecting protein.Complete absorption of protein species by C18 from the conjugate productis shown in profile (1). FIG. 4B provides HPSEC profiles of the samethree injections as in FIG. 4A monitored at 206 nm, detecting proteinand polysaccharide. The peak at 22.5 minutes in post C18 absorption (1)is from the un-absorbed free polysaccharide in the conjugate product.FIG. 4C provides a comparison of HPSEC profile at 206 nm of free PS inconjugate product (1) with those of activated Mn C PS at 0.033 mg/ml(2), 0.067 mg/ml (3), and 0.134 mg/ml (4).

FIG. 5 provides a quantitation of free PS in the Mn C PS-TT conjugateprepared by reductive amination conjugation of aldehyde-activated PS andhydrazide-activated protein. The area of the peak at 22.5 minutes inHPSEC profiles 2, 3 and 4 in FIG. 4C is measured and plotted against itsrespective concentration to construct a standard curve. The content offree PS in conjugate product is calculated from the peak area at 22.5minutes of profile 1 in FIG. 4C.

FIG. 6 provides HPSEC profiles (280 nm) of Mn A PS-TT conjugateMA031219R prepared by reductive amination conjugation ofaldehyde-activated PS and hydrazide-activated protein, and TTH using aWaters Ultrahydrogel Linear column. Upon conjugation, the protein signalshifts from 17.5 minutes to 15 minutes.

FIG. 7 provides HPSEC profiles (280 nm) of Pn 6B PS-TT conjugateprepared by cyanylation conjugation and TTH using a Waters UltrahydrogelLinear column. Upon conjugation, the protein signal shifts from 17minutes to 13.5 minutes. The spectra are taken from conjugation productsbefore a dialysis step and contain extra peaks at greater than 25minutes not seen after dialysis.

FIG. 8 provides HPSEC profiles (280 nm) of Pn 7F PS-TT conjugateprepared by cyanylation conjugation. Upon conjugation, the proteinsignal shifts from 17 minutes to 13.5 minutes. The spectra are takenfrom conjugation products before the dialysis step and contain extrapeaks at greater than 25 minutes not seen after dialysis.

FIG. 9 provides HPSEC profiles (280 nm) of Pn 9V PS-TT conjugateprepared by reductive amination conjugation of hydrazide-activated PSand aldehyde-activated protein TT-aldehyde. Upon conjugation, theprotein signal shifts from 17 minutes to 13.5 minutes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art recognizethat there are numerous variations and modifications of this inventionthat are encompassed by its scope. Accordingly, the description of apreferred embodiment should not be deemed to limit the scope of thepresent invention.

Introduction

Conventional methods for synthesis and manufacturing ofpolysaccharide-protein conjugate vaccines typically employ conjugationreactions with low efficiency (typically about 20%). This means that upto 80% of the added activated polysaccharide is lost. In addition, achromatographic process for purification of the conjugates fromunconjugated PS is typically required. The synthetic methods of thepreferred embodiments utilize the characteristic chemical property ofhydrazide groups on one reactant to react with aldehyde groups orcyanate esters on the other reactant with an improved conjugate yield(typically as high as about 60%).

When the conjugation reaction proceeds with a greater conjugationefficiency, the amount of unconjugated protein and polysaccharideremaining after reaction can be sufficiently low so as to make itsremoval unnecessary. Accordingly, the process of purifying the conjugateproduct can be simplified to, e.g., a diafiltration step for removal ofsmall molecule by-products. The hydrazide-based conjugation reaction canbe carried to completion within one or two days at reactantconcentrations of from about 1 to about 40 mg/mL at PS/protein moleratios of from about 1:5 to about 5:1, preferably from about 1:2 toabout 2:1, and most preferably about 1:1, although in certainembodiments higher or lower ratios can be preferred. The conjugationreaction is preferably conducted at temperatures of from about 4° C. toabout 40° C., preferably from about 5, 10, 15, or 20° C. to about 25,30, or 35° C., and at a pH of from about 6 to about 8.5, preferably fromabout 6.1, 6.2, 6.3, 6.4, or 6.5 to about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4, withoptimal conditions varying according to the polysaccharide. Accordingly,conjugate vaccine can be manufactured at lower cost when ahydrazide-based conjugation reaction is employed.

To overcome certain drawbacks of conventional methods for synthesizingconjugate vaccines, a method for conjugation of PSs to carrier proteinsusing hydrazide chemistry in reduction amination and cyanylationconjugation reactions is provided. Hydrazide groups having the structure—NH—NH₂ are introduced onto the carboxyl groups of the aspartic acidand/or glutamic acid residues of protein molecules by carbodiimidereaction with hydrazine, ADH, carbohydrazide, or succinyl dihydride. Theactivated protein is maintained soluble at a pH of from about 10 toabout 11.5, preferably from about 10.1, 10.2, 10.3, or 10.4 to about10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, or 11.4, and mostpreferably about 10.5, with a buffer at a concentration of from about 3or less to about 10 mM or more, preferably from about 4 or 5 mM to about6, 7, 8, or 9 mM, before conjugation. Suitable buffers include but arenot limited to Na₂CO₃, 3-(cyclohexylamino)-1-propanesulfonicacid (CAPS),and (2-(N-cyclohexylamino)ethane sulfonic acid (CHES). The activatedprotein is then reacted with activated polysaccharide containing eitheraldehyde (reductive amination) or cyanate (cyanylation conjugation)groups at a pH of from about 6 to about 8.5, preferably from about 6.1,6.2, 6.3, 6.4, or 6.5 to about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 in the presence of a buffer at aconcentration about 100 mM or less to about 200 mM, preferably fromabout 110, 120, 130, 140 or 150 mM to about 160, 170, 180 or 190 mM.Suitable buffers include but are not limited to N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), phosphate buffered saline(PBS), TES (EDTA, Tris-HCl, SDS), morpholinopropanesulfonic acid (MOPS),and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES).

Alternatively, the PS can be functionalized with hydrazide groups. Theactivated PS can be conjugated, at pH 6.5-7.5 with a strong buffer, toactivated proteins containing aldehyde groups (reductive amination). Theprotein is maintained soluble at a pH of about 10.5 with a weak bufferuntil the point of conjugation. Because of the higher reactivity ofhydrazide groups (pKa=2.6) compared to the lysine ε-amino group(pKa=10.5) at neutral/mild acidic conditions, and the enhancedsolubility of the conjugate using activated protein maintained solubleat about pH 10.5 before conjugation, the yield of the conjugationreaction is greatly increased. The greater reactivity of thehydrazide-activated tetanus toxoid (TT-H) compared to tetanus toxoid(TT) is illustrated in FIG. 1.

Conjugates prepared by these methods are immunogenic in experimentalanimals, as demonstrated in experiments on mice. In addition, theconjugation reaction can be efficiently carried out without sodiumcyanoborohydride, thereby avoiding introduction of cyanide ion in theconjugate product. The reaction can be conducted under mild acidic orneutral pH conditions at room temperature or at 4° C. overnight asopposed to days for conventional reductive amination conjugationmethods. This again ensures high yield conjugate vaccine production forunstable polysaccharides, such as those from Haemophilus influenzae typeb, Streptococcus pneumoniae type 19F and Neisseria meningitides group A.The methods of preferred embodiments can be employed to produce lessexpensive conjugate vaccines, thereby greatly promoting public health.

The Polysaccharide

“The term “polysaccharide” as used herein, is a broad term and is usedin its ordinary sense, including, without limitation, saccharidescomprising a plurality of repeating units, including, but not limited topolysaccharides having 50 or more repeat units, and oligosaccharideshaving 50 or less repeating units. Typically, polysaccharides have fromabout 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about2,000 or more repeating units, and preferably from about 100, 150, 200,250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 repeating units toabout, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 repeatingunit. Oligosaccharides typically about from about 6, 7, 8, 9, or 10repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45repeating units.

Suitable polysaccharides for use in the preferred embodiments includepolysaccharides and oligosaccharides from encapsulated bacteria. Thepolysaccharides and oligosaccharides can be from any source, forexample, they can be derived from naturally-occurring bacteria,genetically engineered bacteria, or can be produced synthetically. Thepolysaccharides and oligosaccharides can be subjected to one or moreprocessing steps prior to activation, for example, purification,functionalization, depolymerization using mild oxidative conditions,deacetylation, and the like. Post processing steps can also be employed,if desired. Any suitable method known in the art for synthesizing,preparing, and/or purifying suitable polysaccharides andoligosaccharides can be employed.

Polysaccharides and oligosaccharides for use in preferred embodimentsinclude pneumococcal polysaccharides of, for example, serotypes 1, 2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F; meningococcal polysaccharides of serotypes A, B, C,W135, and Y, Haemophilus influenzae type b polysaccharidepolyribosylribitol phosphate, group B streptococcal polysaccharides ofserotypes III and V and Salmonella typhi Vi polysaccharide. Otherpolysaccharides of pneumococcal and group B streptococcal serotypes, andmeningococcal serogroups are also suitable for use herein, as are otherT-independent polysaccharide and oligosaccharide antigens, for example,polysaccharides or oligosaccharides derived from group A streptococcus,Staphylococci, Enterococci, Klebsiella pneumoniae, E. coli, Pseudomonasaeruginosa, and Bacillus anthracis. While bacterial polysaccharides andoligosaccharides are particularly preferred, gram (−) bacteriallipopolysaccharides and lipooligosaccharides and their polysaccharideand oligosaccharide derivatives, and viral polysaccharides andoligosaccharides can also be employed.

Polysaccharides with side chain phosphorus and/or backbone phosphorusare suitable for use in preferred embodiments. The conjugation reactionsof preferred embodiments are particularly well suited for use withpolysaccharides having phosphorus in the backbone. Such polysaccharidesare sensitive to fragmentation and degradation, so the rapidity of theconjugation reaction results in a higher quality conjugate due to thereduced time during which degradation can occur.

After completion of any pre-processing steps, the polysaccharide oroligosaccharide is subjected to an “activation” step. The term“activation” refers to a chemical treatment of the polysaccharide toprovide chemical groups capable of reacting with the protein. In aparticularly preferred embodiment, activation involves functionalizationof the polysaccharide or oligosaccharide with hydrazide groups that arereacted with aldehyde groups on a functionalized protein. Alternatively,the polysaccharide or oligosaccharide can be functionalized withaldehyde groups, ketone groups, or cyanate groups that are reacted withhydrazide groups on a functionalized protein.

Any suitable functionalization reaction can be employed to activate thepolysaccharide or oligosaccharide with hydrazide groups. A preferredfunctionalization reaction is reductive amination, wherein thepolysaccharide or oligosaccharide is reacted with NaIO₄ in a periodateactivation reaction to yield aldehyde groups, which are then reactedwith adipic acid dihydrazide, followed by subsequent reduction withNaBH₄. Alternatively, a cyanylation conjugation reaction can beemployed, wherein polysaccharide or oligosaccharide is reacted withcyanogens bromide or 1-cyano-4-dimethylammoniumpyridiniumtetrafluoroborate to introduce a cyanate group which is subsequentlyreacted with adipic acid dihydrazide. A carbodiimide reaction can alsobe employed, wherein polysaccharide or oligosaccharide is reacted withadipic acid dihydrazide in the presence1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride).

Any suitable functionalization reaction can be employed to activate thepolysaccharide or oligosaccharide with cyanate groups. Preferably, thepolysaccharide or oligosaccharide is reacted with1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate in the presenceof triethylamine.

Any suitable functionalization reaction can be employed to activate thepolysaccharide or oligosaccharide with aldehyde groups. Certainpolysaccharides and oligosaccharides possess terminal aldehyde groupsthat can participate in the conjugation reaction. If the polysaccharideor oligosaccharide is activated with aldehyde groups, a preferredreaction involves reaction with an oxidizing agent, such as NaIO₄.Oxidizing agents have the potential for fragmenting the polysaccharideor oligosaccharide. Undesirable fragmentation can be avoided orcontrolled through selection of the particular oxidizing agent and theconcentration of the oxidizing agent employed. Ketone groups are alsocapable of reacting with hydrazide, so activated of the polysaccharideor oligosaccharide with ketone groups can be employed in certainembodiments.

A strongly buffered (at pH of from about 6.5 to about 8, with a highbuffer concentration of from about 100 mM to about 200 mM) activatedpolysaccharide solution is preferably employed in the conjugationreaction in the form of a strongly buffered solution. Any suitablebuffer can be employed, preferably a buffer such as N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid).

The Protein

The activated polysaccharide or oligosaccharide is coupled to a proteinto yield a conjugate vaccine. Suitable proteins include bacterial toxinsthat are immunologically effective carriers that have been rendered safeby chemical or genetic means for administration to a subject. Examplesinclude inactivated bacterial toxins such as diphtheria toxoid, CRM₁₉₇,tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin Afrom Pseudomonas aeruginosa. Bacterial outer membrane proteins such as,outer membrane complex c (OMPC), porins, transferrin binding proteins,pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesinprotein (PsaA), or pneumococcal surface proteins BVH-3 and BVH-11 canalso be used. Other proteins, such as protective antigen (PA) ofBacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), humanserum albumin, bovine serum albumin (BSA) and purified proteinderivative of tuberculin (PPD) can also be used. The proteins arepreferably proteins that are non-toxic and non-reactogenic andobtainable in sufficient amount and purity that are amenable to theconjugation methods of preferred embodiments. For example, diphtheriatoxin can be purified from cultures of Corynebacteria diphtheriae andchemically detoxified using formaldehyde to yield a suitable protein.

Fragments of the native toxins or toxoids, which contain at least oneT-cell epitope, are also useful, as are outer membrane proteincomplexes, as well as certain analogs, fragments, and/or analogfragments of the various proteins listed above. The proteins can beobtained from natural sources, can be produced by recombinanttechnology, or by synthetic methods as are known in the art. Analogs canbe obtained by various means, for example, certain amino acids can besubstituted for other amino acids in a protein without appreciable lossof interactive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Other proteins can also be employed, such as those containingsurface exposed glutamic acid or aspartic acid groups.

Any suitable functionalization reaction can be employed to activate theprotein with hydrazide groups. Conventional methods for preparinghydrazide-modified proteins include EDC catalysis and a two-step processusing N-succinimidyl iodoacetate and thiol hydrazide through lysines-amino groups of the protein. See King et al., Biochemistry 1986;25:5774-5779. Modified protein prepared by EDC catalysis typically needsto be fractionated in order for it to be suitable for use inconjugation, and the two-step process is tedious. Accordingly, it isgenerally not preferred to employ such methods for preparing thehydrazide-modified protein. However, in certain embodiments such methodscan be acceptable or even desirable.

Preferably, hydrazide groups are introduced into proteins through thecarboxyl groups of aspartic acid and glutamic acid residues on theprotein using a carbodiimide reaction, for example, by reaction withhydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazideor any other dihydrazides in the presence of EDC. EDC is employed as acatalyst to activate and modify the protein reactant with hydrazine orthe dihydrazide. Any water-soluble carbodiimide including EDC can beused as a catalyst. EDC-catalyzed proteins generally have a tendency topolymerize and precipitate, and thus are generally not preferred forpreparation of conjugates involved with protein. See Schneerson et al.,Infect. Immun. 1986, 52:519-528; Shafer et al., Vaccine 2000; 18(13):1273-1281; and Inman et al., Biochemistry 1969; 8:4074-4082. Aggregationand precipitation of the activated protein depends, in part, on its pHenvironment. Accordingly, the tendency to polymerize and precipitate canbe controlled by maintaining such hydrazide-modified proteins soluble ina buffered solution. By buffer-exchanging the reaction mixture so as tomaintain the activated protein at a pH of about 10.5, the activatedprotein remains soluble and stable for conjugation. Any suitable buffercan be employed. Preferably a weak buffer such as Na₂CO₃ at a lowconcentration of from about 3 mM to about 10 mM is employed.

The buffered hydrazide-modified protein can then be employed inpreparing protein-polysaccharide conjugates without precipitation whenadded to activated polysaccharide at a pH of from about 6 to 8.5,preferably from about 6.5 to about 8. Any suitable functionalizationreaction can be employed to activate the protein with aldehyde groups.Preferably, the protein is reacted with 1-amino-2,3-propanediol in thepresence of EDC. Amino sugars such as glucosamine, galactosamine, andthe like can be used in place of 1-amino-2,3-propanediol. In thisreaction, EDC is also employed as a catalyst to activate and modify theprotein reactant with the aminodiol through the carboxyl groups ofaspartic acid and glutamic acid residues of the protein.

Preparation of Conjugates by Reductive Amination

Conjugates can be prepared via the reaction of aldehyde and hydrazidegroups (reductive amination). The reductive amination conjugationreaction can be employed to conjugate a hydrazide-modified reactant(protein or polysaccharide) to the other component containing aldehydegroups.

In conventional reductive amination, the reaction between aldehyde andamino groups is reversible and unfavorable, such that sodiumcyanoborohydride is needed to facilitate the conjugation by convertingthe C═N double bond to a C—N single bond to render the entire reductiveamination event irreversible. In contrast, the reductive aminationconjugation reaction of preferred embodiments proceeds without the aidof sodium cyanoborohydride because of the high efficiency of thehydrazide-aldehyde reaction. At the end of the reductive aminationconjugation reaction, sodium borohydride or another suitable reactant isemployed to reduce the C═N double bond to a C—N single bond, as well asto reduce any residual aldehyde groups to alcohol groups. The reductiveamination conjugation reaction of preferred embodiments avoidscontamination of the resulting conjugate with cyanide, a by-product ofsodium cyanoborohydride.

To reduce precipitation of activated protein during the conjugationreaction, the activated protein is preferably in the form of a weaklybuffered solution with a low buffer concentration of from about 3 mM toabout 10 mM which is added to a strongly buffered (at pH of from about6.5 to about 7.5, with a high buffer concentration of from about 100 mMto about 200 mM) activated polysaccharide solution. Preferably, the pHof the activated protein solution is buffered to from about 10 pH toabout 11.5 pH, most preferably to about 10.5 pH. The activatedpolysaccharide solution is preferably strongly buffered to from about 6pH to about 8 pH, most preferably to from about 6.5 pH to about 7.5 pH.The hydrazide-aldehyde reductive amination reaction proceeds at a fastrate, and the precipitating effect of a pH lower than 10.5 (for example,a pH as low as from about 8.5 to about 9.5) on activated protein isovercome by the molecular properties of the reacting activatedpolysaccharide.

Preparation of Conjugates by Cyanylation Conjugation

Conjugates can be prepared via the reaction of hydrazide and cyanategroups (cyanalation conjugation). The cyanalation conjugation reactionis efficient and reversible, favoring the product formation. In certainembodiments, blocking agents are employed to remove residual cyanategroups. However, addition of a blocking agent to the reaction mixturedrives the conjugation reaction backward and reduces the conjugationyield by 5-12%. The effect of various blocking agents on yield wasinvestigated. The pneumococcal polysaccharide Pn 18C PS was activatedwith CDAP and then conjugated to hydrazide activated tetanus toxoid(TTH) overnight. Five aliquots were added with either water or ablocking agent to 0.2 M. After 4 hours incubation, the samples wereanalyzed by HPSEC using a Waters Ultrahydrogel 2000 column with a 280 nmmonitor (FIG. 2). The conjugation yield of each sample, provided inTable 3, was determined as the % area of the conjugate peak at 15.5minutes over total protein, i.e. conjugate peak plus the free TTH peak(at 22 minutes). While in certain embodiments it can be desirable toemploy blocking agents to quench the leftover residual cyanate groups,it is generally preferred to avoid their use so as to avoid reduction inconjugate yield.

TABLE 3 Blocking agent (0.2M) Conjugation yield % Control % ReductionNone (control) 75 100 0 ADH 63 84 16 Hydrazine 70 93 7 Glycine 66 89 11Ethanolamine 65 87 13

To remove residual cyanate groups in the conjugation product withoutusing a blocking agent, the conjugation time can be prolonged.Preferably, conjugation is conducted at a temperature of from about 0°C. to about 5° C. for about 36 to about 48 hours, most preferably atabout 4° C. for about 36 hours, followed by about an additional 18 to 24hours at a temperature of from about 20° C. to about 25° C., mostpreferably at about 18 hours at about 20 to 24° C., such that theresidual cyanate groups react with water and decompose. Longer orshorter conjugation times and/or higher or lower conjugationtemperatures can be employed, and different sequences of steps atvarious times and temperatures can be conducted, as desired. It isdesirable, however, to conduct the conjugation reaction, at leastinitially, at low temperatures, preferably from about 0° C. to about 5°C., more preferably at about 4° C., so as to reduce the degree ofprecipitation of the conjugate.

With high conjugation yields and high immunogenicity of the conjugationproduct, purification processes such as column chromatography and/orammonium sulfate precipitation of the conjugate from unconjugatedpolysaccharide may not be necessary. However, in certain embodiments itcan be desirable to conduct one or more purification steps.

The Conjugates

Both reactants contain multiple reactive groups per molecule. Anactivated polysaccharide molecule can react with and form more than onelinkage to more than one activated protein molecule. Likewise, anactivated protein molecule can react with and form more than one linkageto more than one activated polysaccharide molecule. Therefore, theconjugate product is a mixture of various crosslinked matrix-typelattice structures. For example, a single linkage can be present, or 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 or more linkages can be present. Theaverage number of linkages between a polysaccharide and a protein can beadjusted, as preferred. The preferred average number of linkages candepend upon the type of polysaccharide, the type of protein, theconjugation method, the reaction conditions, and the like. Generally, anaverage of 1 linkage to about 2, 3, 4, or 5 linkages is present, so asto avoid interfering with the ability of the protein to stimulate theimmune system by over-conjugation, and so as to not cause changes in thepolysaccharide structure. However, in certain embodiments more than 5linkages can be tolerated or even desirable.

After conjugation, the conjugate can be purified by any suitable method.Purification is employed to remove unreacted polysaccharide, protein, orsmall molecule reaction byproducts. Purification methods includeultrafiltration, size exclusion chromatography, density gradientcentrifugation, hydrophobic interaction chromatography, ammonium sulfatefractionation, and the like, as are known in the art. As discussedabove, the conjugation reactions of preferred embodiments proceed withhigher yield, and generate fewer undesirable small molecule reactionbyproducts. Accordingly, no purification may be necessary, or only aminor degree of purification can be desirable. The conjugate can beconcentrated or diluted, or processed into any suitable form for use inpharmaceutical compositions, as desired.

Methods of Treatment

Conjugates prepared according to the preferred embodiment areadministered to a subject in an immunologically effective dose in asuitable form to treat and/or prevent infectious diseases. The term“subject” as used herein, refers to animals, such as mammals. Forexample, mammals contemplated include humans, primates, dogs, cats,sheep, cattle, goats, pigs, horses, mice, rats, rabbits, guinea pigs,and the like. The terms “subject”, “patient”, and “host” are usedinterchangeably. As used herein, an “immunologically effective” dose ofthe conjugate vaccine is a dose which is suitable to elicit an immuneresponse. The particular dosage depends upon the age, weight and medicalcondition of the subject to be treated, as well as on the method ofadministration. Suitable doses can be readily determined by those ofskill in the art.

Pharmaceutical compositions comprising conjugate vaccines of preferredembodiments can offer various advantages over conventional vaccines,including enhanced immunogenicity of weakly immunogenic antigens,potential reduction in the amount of antigen used, less frequent boosterimmunizations, improved efficacy, preferential stimulation of immunity,or potential targeting of immune responses. The vaccines can beadministered to a subject by a variety of routes, as discussed below,including but not limited to parenteral (e.g., by intracisternalinjection and infusion techniques), intradermal, transmembranal,transdermal (including topical), intramuscular, intraperitoneal,intravenous, intra-arterial, intralesional, subcutaneous, oral, andintranasal (e.g., inhalation) routes of administration. Conjugatevaccine can be administered by bolus injection or by continuousinfusion, as well as by localized administration, e.g., at a site ofdisease or injury. The conjugate vaccine can be optionally administeredin a pharmaceutically or physiologically acceptable vehicle.

The term “vaccine” as used herein, is a broad term and is used in itsordinary sense, including, without limitation, conjugates of preferredembodiments or other antigens formulated with adjuvants, diluents,excipients, carriers, and other pharmaceutically acceptable substances.The term “pharmaceutically acceptable” is used to refer to a non-toxicmaterial that is compatible with a biological system such as a cell,cell culture, tissue, or organism.

Immunization protocols for use with the conjugates of preferredembodiments provide compositions and methods for preventing or treatinga disease, disorder and/or infection in a subject. The term “treating”as used herein, is a broad term and is used in its ordinary sense,including, without limitation, curative, preventative, prophylactic,palliative and/or ameliorative treatment.

The vaccine compositions are preferably sterile and contain either atherapeutically or prophylactically effective amount of the conjugate ina unit of weight or volume suitable for administration to a subject. Theterm “pharmaceutically-acceptable carrier” as used herein means one ormore compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration into a subject. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The characteristics of the carrier depend on the routeof administration. Physiologically and pharmaceutically-acceptablecarriers include diluents, fillers, salts, buffers, stabilizers,solubilizers, and other materials which are well known in the art.

The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the conjugates of the preferred embodiment, andwith each other, in a manner such that there is no interaction whichsubstantially impairs the desired pharmaceutical efficacy.

Formulation of the conjugate vaccines of preferred embodiments intopharmaceutical compositions can be accomplished using methods known inthe art. The vaccine compositions can also contain one or moreadjuvants. Suitable adjuvants include, for example, aluminum adjuvants,such as aluminum hydroxide or aluminum phosphate, Freund's Adjuvant,BAY, DC-chol, pcpp, monophoshoryl lipid A, CpG, QS-21, cholera toxin andformyl methionyl peptide. See, e.g., Vaccine Design, the Subunit andAdjuvant Approach, 1995 (M. F. Powell and M. J. Newman, eds., PlenumPress, N.Y.).

The dosage of conjugate vaccine to be administered a subject and theregime of administration can be determined in accordance with standardtechniques well known to those of ordinary skill in the pharmaceuticaland veterinary arts, taking into consideration such factors as theintended use, particular antigen, the adjuvant (if present), the age,sex, weight, species, general condition, prior illness and/ortreatments, and the route of administration. Preliminary doses can bedetermined according to animal tests, and the scaling of dosages forhuman administration is performed according to art-accepted practicessuch as standard dosing trials. For example, the therapeuticallyeffective dose can be estimated initially from serum antibody leveltesting. The dosage depends on the specific activity of the conjugateand can be readily determined by routine experimentation.

In practicing immunization protocols for treatment and/or prevention ofspecified diseases, a therapeutically effective amount of conjugate isadministered to a subject. As used herein, the term “effective amount”means the total amount of therapeutic agent (e.g., conjugate) or otheractive component that is sufficient to show a meaningful benefit to thesubject, such as, enhanced immune response, treatment, healing,prevention or amelioration of the relevant medical condition (disease,infection, or the like), or an increase in rate of treatment, healing,prevention or amelioration of such conditions. When “effective amount”is applied to an individual therapeutic agent administered alone, theterm refers to that therapeutic agent alone. When applied to acombination, the term refers to combined amounts of the ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. As used herein, the phrase “administering aneffective amount” of a therapeutic agent means that the subject istreated with said therapeutic agent(s) in an amount and for a timesufficient to induce an improvement, and preferably a sustainedimprovement, in at least one indicator that reflects the severity of thedisease, infection, or disorder.

An improvement is considered “sustained” if the patient exhibits theimprovement on at least two occasions separated by a period of time. Thedegree of improvement can be determined based, for example, onimmunological data, or on signs or symptoms of a disease, infection, ordisorder. Various indicators that reflect the extent of the patient'sillness can be assessed for determining whether the amount and time ofthe treatment is sufficient. The baseline value for the chosen indicatoror indicators can established based on by examination of the patientprior to administration of the first dose of the therapeutic agent, orbased on statistical values generated from a population of healthypatients. If the therapeutic agent is administered to treat acutesymptoms, the first dose is administered as soon as practicallypossible. Improvement is induced by administering therapeutic agentsuntil the subject manifests an improvement over baseline for the chosenindicator or indicators. In treating chronic conditions, this degree ofimprovement is obtained by repeatedly administering the therapeuticagents over a period time, e.g., for one, two, or three months orlonger, or indefinitely. A single dose can be sufficient for treating orpreventing certain conditions. Treatment can be continued indefinitelyat the same level or at a reduced dose or frequency, regardless of thepatient's condition, if desired. Once treatment has been reduced ordiscontinued, it later can be resumed at the original level if symptomsreappear.

Generally, the amount of conjugate that provides an efficacious dose ortherapeutically effective dose for vaccination against bacterialinfection is from about 1 μg or less to about 100 μg or more, preferablyfrom about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50μg to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 μg per kg body weight.An efficacious dosage can require less antibody if the post-infectiontime elapsed is less, since there is less time for the bacteria toproliferate. An efficacious dosage can also depend on the bacterial loadat the time of diagnosis. Multiple injections administered over a periodof days can be considered for therapeutic usage.

The conjugate vaccines can be administered as a single dose or in aseries including one or more boosters. For example, an infant or childcan receive a single dose early in life, then be administered a boosterdose up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years later. Thebooster dose generates antibodies from primed B-cells, i.e., ananamnestic response. That is, the conjugate vaccine elicits a highprimary functional antibody response in infants or children, and iscapable of eliciting an anamnestic response following a boosteradministration, demonstrating that the protective immune responseelicited by the conjugate vaccine is long-lived.

The conjugate vaccines can be formulated into liquid preparations for,e.g., oral, nasal, anal, rectal, buccal, vaginal, peroral, intragastric,mucosal, perlinqual, alveolar, gingival, olfactory, or respiratorymucosa administration. Suitable forms for such administration includesuspensions, syrups, and elixirs. The conjugate vaccines can also beformulated for parenteral, subcutaneous, intradermal, intramuscular,intraperitoneal or intravenous administration, injectableadministration, sustained release from implants, or administration byeye drops. Suitable forms for such administration include sterilesuspensions and emulsions. Such conjugate vaccines can be in admixturewith a suitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, and the like. The conjugate vaccines canalso be lyophilized. The conjugate vaccines can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavoringagents, colors, and the like, depending upon the route of administrationand the preparation desired. Standard texts, such as “Remington: TheScience and Practice of Pharmacy”, Lippincott Williams & Wilkins; 20thedition (Jun. 1, 2003) and “Remington's Pharmaceutical Sciences”, MackPub. Co.; 18^(th) and 19^(th) editions (December 1985, and June 1990,respectively), incorporated herein by reference in their entirety, canbe consulted to prepare suitable preparations, without undueexperimentation Such preparations can include complexing agents, metalions, polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, and the like, liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vive release, and rate of in vive clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

The conjugate vaccines are preferably provided as liquid suspensions oras freeze-dried products. Suitable liquid preparations include, e.g.,isotonic aqueous solutions, suspensions, emulsions, or viscouscompositions that are buffered to a selected pH. Transdermalpreparations include lotions, gels, sprays, ointments or other suitabletechniques. If nasal or respiratory (mucosal) administration is desired(e.g., aerosol inhalation or insufflation), compositions can be in aform and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or adose having a particular particle size, as discussed below.

When in the form of solutions, suspensions and gels, formulations of theconjugate can typically contain a major amount of water (preferablypurified water) in addition to the active ingredient. Minor amounts ofother ingredients such as pH adjusters, emulsifiers, dispersing agents,buffering agents, preservatives, wetting agents, jelling agents, colors,and the like can also be present.

The compositions are preferably isotonic with the blood or other bodyfluid of the recipient. The isotonicity of the compositions can beattained using sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is particularly preferred. Bufferingagents can be employed, such as acetic acid and salts, citric acid andsalts, boric acid and salts, and phosphoric acid and salts. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride, lactated Ringer's or fixed oils. Intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.

Viscosity of the compositions can be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener can dependupon the agent selected. The important point is to use an amount thatcan achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the compositions. Benzyl alcohol can be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride can also beemployed. A suitable concentration of the preservative can be from 0.02%to 2% based on the total weight although there can be appreciablevariation depending upon the agent selected.

Pulmonary delivery of the conjugate can also be employed. The conjugateis delivered to the lungs of a mammal while inhaling and traversesacross the lung epithelial lining to the blood stream. A wide range ofmechanical devices designed for pulmonary delivery of therapeuticproducts can be employed, including but not limited to nebulizers,metered dose inhalers, and powder inhalers, all of which are familiar tothose skilled in the art. These devices employ formulations suitable forthe dispensing of the conjugate. Typically, each formulation is specificto the type of device employed and can involve the use of an appropriatepropellant material, in addition to diluents, adjuvants and/or carriersuseful in therapy.

The conjugate is advantageously prepared for pulmonary delivery inparticulate form with an average particle size of from 0.1 μm or less to10 μm or more, more preferably from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, or 0.9 μm to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 μm for pulmonarydelivery. Pharmaceutically acceptable carriers for pulmonary delivery ofthe conjugates include carbohydrates such as trehalose, mannitol,xylitol, sucrose, lactose, and sorbitol. Other ingredients for use informulations can include DPPC, DOPE, DSPC and DOPC. Natural or syntheticsurfactants can be used, including polyethylene glycol and dextrans,such as cyclodextran. Bile salts and other related enhancers, as well ascellulose and cellulose derivatives, and amino acids can also be used.Liposomes, microcapsules, microspheres, inclusion complexes, and othertypes of carriers can also be employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, typically comprise the conjugate dissolved or suspended inwater at a concentration of about 0.01 or less to 100 mg or more ofconjugate per mL of solution, preferably from about 0.1, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90 mg of conjugate per mL of solution. Theformulation can also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the conjugate caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such chlorofluorocarbon, ahydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons, such astrichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, andcombinations thereof. Suitable surfactants include sorbitan trioleate,soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing the conjugate,optionally including a bulking agent, such as lactose, sorbitol,sucrose, mannitol, trehalose, or xylitol in an amount that facilitatesdispersal of the powder from the device, typically from about 1 wt. % orless to 99 wt. % or more of the formulation, preferably from about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75,80, 85, or 90 wt. % of the formulation.

When the conjugate is administered by intravenous, cutaneous,subcutaneous, or other injection, the conjugate vaccine is preferably inthe form of a pyrogen-free, parenterally acceptable aqueous solution.The preparation of parenterally acceptable solutions with suitable pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred pharmaceutical composition for injection preferably containsan isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicles as are known in the art.The pharmaceutical compositions can also contain stabilizers,preservatives, buffers, antioxidants, or other additives known to thoseof skill in the art.

The duration of the injection can vary depending upon various factors,and can comprise a single injection administered over the course of afew seconds or less, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuousintravenous administration.

The conjugate can be administered topically, systematically, or locally,via a liquid or gel, or as an implant or device

The conjugates of preferred embodiments, or the conjugation methods ofpreferred embodiments, can be useful in preparing vaccines for thetreatment of a variety of bacterial infections, including infections byHelicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps. (e.g. M. tubercilosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, and Actinomyces israelli.

Certain methods of the preferred embodiments can also be of use inpreparing vaccines for treating or vaccinating subjects against cancer,such as mammalian sarcomas and carcinomas, such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,serminoma, embryonal carcinoma, Wilms' tumor, cervical cancer,testicular tumor, lung carcinoma, small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, such as acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease, lymphoproliferative disorders including autoimmunelymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia,hairy cell leukemia, chronic lymphatic leukemia, peripheral T-celllymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicularlymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T celllymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressivelymphoma, acute lymphatic leukemias, T gamma lymphoproliferativedisease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (i.e.,mycosis fungoides) and Szary syndrome.

The conjugates can be administered in combination with various vaccineseither currently being used or in development, whether intended forhuman or non-human subjects. Examples of vaccines for human subjects anddirected to infectious diseases include the combined diphtheria andtetanus toxoids vaccine; pertussis whole cell vaccine; the inactivatedinfluenza vaccine; the 23-valent pneumococcal vaccine; the live measlesvaccine; the live mumps vaccine; live rubella vaccine; BacilleCalmette-Guerin (BCG) tuberculosis vaccine; hepatitis A vaccine;hepatitis B vaccine; hepatitis C vaccine; rabies vaccine (e.g., humandiploid cell vaccine); inactivated polio vaccine; meningococcalpolysaccharide vaccine; quadrivalent meningococcal vaccine; yellow feverlive virus vaccine; typhoid killed whole cell vaccine; cholera vaccine;Japanese B encephalitis killed virus vaccine; adenovirus vaccine;cytomegalovirus vaccine; rotavirus vaccine; varicella vaccine; anthraxvaccine; small pox vaccine; and other commercially available andexperimental vaccines.

The conjugates can be provided to an administering physician or otherhealth care professional in the form of a kit. The kit is a packagewhich houses a container which contains the conjugate vaccine andinstructions for administering the conjugate vaccine to a subject. Thekit can optionally also contain one or more other therapeutic agents.The kit can optionally contain one or more diagnostic tools andinstructions for use. For example, a vaccine cocktail containing two ormore vaccines can be included, or separate pharmaceutical compositionscontaining different vaccines or therapeutic agents. The kit can alsocontain separate doses of the conjugate vaccine for serial or sequentialadministration. The kit can contain suitable delivery devices, e.g.,syringes, inhalation devices, and the like, along with instructions foradministrating the therapeutic agents. The kit can optionally containinstructions for storage, reconstitution (if applicable), andadministration of any or all therapeutic agents included. The kits caninclude a plurality of containers reflecting the number ofadministrations to be given to a subject. If the kit contains a firstand second container, then a plurality of these can be present.

EXPERIMENTS

Materials

Tetanus toxoid (TT) was obtained from Lederle Vaccines, Pearl River,N.Y. and Serum Institute of India, Pune, India. Meningococcal groups Aand C polysaccharides (Mn A PS and Mn C PS, respectively) were obtainedfrom Bio-Manguinhos, Rio de Janeiro, Brazil. Mn A PS was also obtainedfrom SynCo Bio Partners, Amsterdam, The Netherlands. Mn W135 and Y PS'swere obtained from Aventis Pasteur. PSs of Pneumococcus (Pn) serotypes1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,19F, 20, 22F, 23F and 33F were obtained from Lederle Vaccines. PS ofHemophilus influenzae type b (PRP or Hib PS) was obtained from LederleVaccines. Vi PS of Salmonnella typhi was obtained from Aventis Pasteur.PSs of group B streptococcus serotypes III and V were isolated fromculture media according to the published protocol. See Carey et al.,Infection and Immunity 1980; 28:195-203. Hydrazine, carbohydrazide,adipic acid dihydrazide (ADH), 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC), N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), sodium periodate, sodiumborohydride, sodium cynoborohydride, 4-cyno-dimethylamino pyridiumtetrafluoroborate (CDAP), and 1-amino-2,3-propanediol were purchasedfrom Sigma/Aldrich Chemical Company. TNBSA(2,4,6-trinitrobenzenesulfonic acid) and BCA (bicinchoninic acid) assaykit were purchased from Pierce.

Methods

The bacterial polysaccharides used for conjugation to protein by themethods described herein include Meningococcal serogroups A, C, W135 andY polysaccharides, Pneumococcus serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N,9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33Fpolysaccharides, Hemophilus influenzae type b polysaccharide (PRP or HibPS), Vi polysaccharide of Salmonnella typhi and group B Streptococcusserotypes III and V polysaccharides. Three general methods are describedfor conjugating polysaccharides to protein, referred to below as GeneralMethod A, General Method B, and General Method C.

General Method A: Aldehyde-Activated PS to Hydrazide-Activated Protein(Reductive Amination Conjugation)

Tetanus toxoid is activated with hydrazine or adipic acid dihydrazide inthe presence of EDC at pH 6.5 and then buffer exchanged with 30 mM NaCl,3 mM Na₂CO₃, pH about 10.5. Polysaccharide is activated with NaIO₄, andbuffer exchanged with 10 mM HEPES, pH 7.5, 4° C. Hydrazide-activated TTis reacted with aldehyde-activated polysaccharide at ratios from 2:1 to1:2 and concentration range of 1-40 mg/mL overnight, pH 6.5-7.5, 4-40°C. NaBH₄ (ten-fold moles of the aldehyde groups in the initial reactant)is then added for 6 hrs to reduce the C═N double bond to C—N single bondand also reduce the unreacted aldehyde groups to alcohol. The solutionis buffer-exchanged with saline, 10 mM HEPES, pH 7.5, 1 mM EDTA using a12-14 KDa molecular weight cut-off membrane. The total protein contentis determined by Lowry assay (see Pierce Catalog 2003-2004, page 306).Total polysaccharide content is determined by various chemical assaysfor different bacterial polysaccharides, e.g. resorcinol assay for Mn Aand C PSs (Monsigny et al., Anal. Biochem. 1988; 175:525-530), anthroneassay for Pn PSs (Keleti et al., Handbook of micromethods for thebiological sciences. 61. Hexoses (Anthrone). Page 73. Van NostrandReinhold Co., New York, 1974), phosphorus assay for Mn A PS, Hib PS andphosphorus-containing Pn PS, and purpald assay for Mn W135 and Y PSs andglycol-containing Pn PSs (Lee et al., Anal. Biochem. 2001; 296:73-82).

General Method B: Cyanate-Activated PS to Hydrazide-Activated Protein(Cyanylation Conjugation)

Tetanus toxoid is activated with hydrazine or adipic acid dihydrazide inthe presence of EDC at pH 6.5 and then buffer exchanged with 30 mM NaCl,3 mM Na₂CO₃, pH about 10.5. Polysaccharide is activated with CDAP for2-2.5 minutes at 20-24° C. in the presence of triethylamine. At 4° C.,hydrazide-activated TT is reacted with cyanate-activated polysaccharideat ratios from 2:1 to 1:2 and concentration range of 0.2-1 mg/mL, pH6.5-7.5. After reaction for 36 hours at 4° C., the mixture is incubatedat 20-24° C. for another 18 hours. The prolonged incubation is to ensuredecomposition of the residual leftover unreacted cyanate groups. Thesolution is buffer-exchanged with saline, 10 mM HEPES, pH 7.5, 1 mM EDTAusing a 12-14 KDa molecular weight cut-off membrane. The total proteincontent is determined by Lowry assay, as noted above in reference toGeneral Method A. Total polysaccharide content is determined by variouschemical assays for different bacterial polysaccharides, e.g.,resorcinol assay for Mn A and C PSs, anthrone assay for Pn PSs,phosphorus assay for Mn A PS, Hib PS and phosphorus-containing Pn PS,and purpald assay for Mn W135 and Y PSs and glycol-containing Pn PSs, asnoted above in reference to General Method A.

General Method C: Hydrazide-Activated PS to Aldehyde-Activated Protein(Reductive Amination Conjugation)

Tetanus toxoid is reacted with 1-amino-2,3-propanediol (APDO) in thepresence of EDC at pH 6.5 and then buffer-exchanged with 30 mM NaCl, 3mM Na₂CO₃, pH about 10.5. TT-APDO is reacted with NaIO₄ to createaldehyde groups and then buffer exchanged with 30 mM NaCl, 3 mM Na₂CO₃,pH about 10.5. Three methods used to prepare hydrazide-activatedpolysaccharide: a) PS is reacted with NaIO₄ and then adipic aciddihydrazide with subsequent reduction with NaBH₄ (reductive amination);b) PS is activated with CDAP and then reacts with adipic aciddihydrazide (cyanylation conjugation reaction); or c) Ps is reacted withadipic acid dihydrazide in the presence EDC (carbodiimide reaction).Aldehyde-activated TT is reacted with hydrazide-activated PS at ratiofrom 2:1 to 1:2 and concentration range 1-5 mg/mL for 18 hours, pH6.5-7.5, 4-40° C. NaBH₄ (ten-fold moles of the aldehyde in the initialreactant) is then added for 6 hrs to reduce the C═N double bond to C—Nsingle bond and also reduce the unreacted aldehyde groups to alcohol.The solution is buffer-exchanged with saline, 10 mM HEPES, pH 7.5, 1 mMEDTA using a 12-14 KDa molecular weight cut-off membrane. The totalprotein content is determined by Lowry assay, as noted above inreference to General Method A. Total polysaccharide content isdetermined by various chemical assays for different bacterialpolysaccharides, e.g., resorcinol assay for Mn A and C PSs, anthroneassay for Pn PSs, phosphorus assay for Mn A PS, Hib PS andphosphorus-containing Pn PS, and purpald assay for Mn W135 and Y PSs andglycol-containing Pn PSs, as noted above in reference to General MethodA.

Physico-Chemical Assays of Reactants, Activated PS and ConjugateProducts

High Performance Liquid Size-Exclusion Chromatography (HPSEC)

Samples of proteins, polysaccharides and conjugate products (25 μL,0.01-1 mg/mL) were run through a Waters Ultrahydrogel 2000 orUltrahydrogel Linear column with saline at 0.5 mL/minute in a DionexHPLC system using Chromelean software, a UV detector at 280 and 206 nmand a Waters 2410 differential refractometer (RI detector). The UVdetector at 280 nm monitored the signals of protein-containing speciesas well as compounds containing aromatic moieties. The UV detector at206 nm detected the protein and PS by presence of carbonyl groups, whilethe RI detector measured the signals of proteins, polysaccharides,conjugates and salts.

Immunogenicity of Polysaccharide-Protein Conjugates in Mice

Immunization of Mice

Mice (NIH-Swiss; groups of 10) were immunized with 1 μg/dose ofpolysaccharide or polysaccharide-protein conjugate prepared by GeneralMethod A (Mn A PS-TT and Mn C PS-TT conjugates), B (Pn 6B PS-TT and Pn7F PS-TT conjugates) and General Method C (Pn 9V PS-TT conjugate) ondays 0 and 14, or on days 0, 14 and 28, with antisera collected on day28 and 42, respectively. ELISA was carried out for determination ofantibody levels against respective native polysaccharides.

ELISA Method

Immunolon 1 plates (Dynatech) were coated with 100 μL coating solutioncontaining polysaccharide (5 μg/mL for Mn A and C, and Pn 7F PS's; 2μg/mL for Pn 6B PS; and 2.5 μg/mL for Pn 9V PS) admixed with methylatedhuman serum albumin (5 μg/mL for Mn A and C, and Pn 6B and 7F PS's; and2.5 μg/mL for Pn 9V PS) for 18 hours. After washing three times with 150μL washing buffer (PBS with 0.05% Tween 20, 0.02% NaN₃), 100 μL ofspecific anti-serum samples and reference serum (with assigned 3200units/mL anti-polysaccharide antibody; duplicate) at a serial two-folddilution starting from 1/200 (diluted with dilution buffer containingPBS, 4% newborn calf serum, 0.02% NaN₃ (with 2 μg/mL cell wallpolysaccharide in pneumococcal cases)), was added to each well. Afterovernight incubation, the plates were washed three times and incubatedwith 100 μL goat anti-mouse IgG Fe conjugated with alkaline phosphate(1/3000 dilution in dilution buffer) for two hours. After washing (3×150μL) the plates were incubated with 100 μL p-nitrophenyl phosphate (1mg/mL in 1 M Tris, pH 9.8, 0.3 mM MgCl₂) for 30 minutes and the reactionwas stopped by 50 μL 1 N NaOH. The ELISA readings were measured with aplate reader and the anti-polysaccharide antibody levels of theantiserum samples were calculated from the ELISA readings and thestandard curve of the reference serum co-assayed in the same plate. Thegeometric mean of antibody level for each mouse group was calculated.

SPECIFIC EXAMPLES Method A Meningococcus Group C Conjugate

Activation of TT to Contain Hydrazide Groups

Tetanus toxoid (4.2 mg/mL) was activated with 0.42 M hydrazine or adipicacid dihydrazide in the presence of 20 mM EDC, 0.1 M MES, pH 6.5 at20-24° C. After reacting for 4 hours, the pH of the reaction mixture wasraised to 7.5-10 with 1 N NaOH to stop the reaction. The reactionmixture was buffer-exchanged with 30 mM NaCl, 3 mM Na₂CO₃, pH about 10.5at 4° C. using a 12-14 KDa dialysis membrane. The protein concentrationof the resulting TT-hydrazide sample was determined by Lowry assay (seePierce Catalog 2003-2004, page 306) using bovine serum albumin as astandard. The hydrazide content was determined by TNBS assay usingadipic acid dihydrazide as a standard, as described in Vidal, J.Immunol. Methods 1986; 86:155-156. The degree of activation of TT soprepared was approximately 50 hydrazide groups per TT molecule.

Activation of Mn C PS to Contain Aldehyde Groups

Mn C PS (10 mg/mL) was reacted with 6 mM NaIO₄ at 20-24° C. for 4 hours.The sample was dialyzed against 10 mM HEPES, pH 7.5 at 4° C. using a12-14 KDa dialysis membrane. The concentration of the resultingactivated PS was determined by resorcinol assay using N-acetylneuraminic acid as the standard with a correction factor of Mn CPS/N-acetyl neuraminic acid=1.104/1, as described in Monsigny et al.,Anal. Biochem. 1988; 175:525-530. The aldehyde content of the activatedPS was determined by BCA (Pierce Catalog 2003-2004, pages 241 and 305)assay using glucose as a standard. The degree of activation of theactivated Mn C PS prepared by this protocol was approximately onealdehyde group per 80 monomers.

Conjugation of Activated Mn C PS to Activated TT

An aliquot of hydrazide-containing TT was adjusted to 25 mg/mL bylyophilization and dissolution in water. An aliquot ofaldehyde-containing Mn C PS was adjusted to 25 mg/mL by lyophilizationand dissolution in 0.2 M HEPES, pH 7.5, 30 mM EDTA. The activated TTsolution was added to an equal volume of the activated Mn C PS andvortexed. The reaction mixture was incubated at 20-24° C. for 18 hours.The reaction mixture was treated with NaBH₄ (10-fold molar equivalent toinitial aldehyde concentration in the activated PS) for 6 hours. Thesolution was buffer-exchanged with saline, 10 mM HEPES, pH 7.5, 1 mMEDTA using a 12-14 KDa molecular weight cut-off membrane. Total proteinwas determined by Lowry assay using bovine serum albumin as a standard.Total Mn C PS content was determined by resorcinol assay using N-acetylneuraminic acid as a standard, as described in Monsigny et al., Anal.Biochem. 1988; 175:525-530.

Four Mn C PS-TT conjugates were prepared using hydrazine or adipic aciddihydrazide as a spacer. FIG. 3 shows the HPSEC elution profiles(monitored at 280 nm) of these conjugates. A slight shift among theprofiles and a right shoulder at 22.5-25 minutes of unconjugated freeprotein were observed.

The unconjugated free Mn C PS was determined by the method of C18particle absorption of protein in the conjugate product followed bycomparing the saccharide signal of the supernatant in HPSEC to those ofthe activated Mn C PS of known concentrations (FIGS. 4 and 5). Toestimate the yield of the conjugation reaction, the conjugate productwas diluted to approximately 1 mg/mL concentration of Mn C PS. 100 μL ofthis solution was mixed and incubated with 250 μL of activated C18particles for an hour with gentle agitation. The supernatant wascollected after centrifugation, and the C18 gel was washed twice with100 μL saline. The combined supernatant and wash was adjusted to 333 μLwith saline and passed through a 0.2 un membrane microfilter. Thefiltrate was analyzed with HPSEC together with standard concentrationsof activated Mn C PS at 0.033, 0.067 and 0.134 mg/mL, giving the area ofthe saccharide signals of these samples as 19.4, 4.8, 9.2, and 18.4,respectively. The saccharide concentration of the filtrate wascalculated from the standard curve as 0.141 mg/mL, which was 3.3 timesvolume of the starting sample. Thus the starting sample contained 0.465mg/mL (0.141 mg/mL×3.3) free Mn C PS. The total Mn C PS concentrationwas determined as 1.131 mg/mL by modified resorcinol assay. The yieldwas estimated to be about 60% (100%×(1−0.465/1.131)).

Immunogenicity of Mn C PS-TT Conjugates

The conjugates prepared as described above were used to immunize groupsof 10 mice with native polysaccharide as a control at 1 Mgpolysaccharide/dose on days 0 and 14. The geometric means of the inducedantibody levels (units/mL) two weeks post 2^(nd) injection were 16 (8,34; 1 SD confidence interval) for control group and 2141 (1069, 4285),4228 (2189, 8167), 1092 (655, 1820) and 3977 (2423, 6526) for the fourconjugate batches made by Method A, assuming 3200 units/mL for thereference serum (Table 4). The conjugates induced 68-264 fold moreanti-Mn C PS specific antibody in mice as compared to the native Mn C PScontrol.

TABLE 4 Mouse groups of different Spacer Geometric Fold conjugate andused mean anti-MCPS increase polysaccharide in the antibody level, overcontrol immunogens conjugate units/mL (CI)^(a) group Native Mn C PS — 16(8, 34)   — (control) Conjugate MC6xTTH Hydrazine 2141 (1096, 4285) 134Conjugate Mix TTHb Hydrazine 4228 (2189, 8167) 264 Conjugate MC6xTTADHADH 1092 (655, 1820)   68 Conjugate Mix bTTADH ADH 3977 (2423, 6526) 248^(a)The geometric mean anti-Mn C PS antibody levels (compared to areference serum with anti-Mn C PS antibody level of 3200 units/mL) with1 SD confidence interval of mouse groups (10 mice per group) two weekspost 2^(nd) immunization with 1 μg/dose native Mn C PS or each of thefour Mn C PS-TT conjugates.

Method A Meningococcus Group a Conjugate

Activation of TT to Contain Hydrazide Groups

Tetanus toxoid (4.2 mg/mL) is activated with 0.42 M hydrazine in thepresence of 20 mM EDC, 0.1 M MES, pH 6.5 at 20-24° C. After reaction for4 hours, the pH of the reaction mixture was raised to 7.5-10 with 1 NNaOH to stop the reaction. The reaction mixture is buffer-exchanged with30 mM NaCl, 3 mM Na₂CO₃, pH about 10.5 at 4° C. using a 12-14 KDadialysis membrane. The protein concentration of the resultingTT-hydrazide sample was determined by Lowry assay (see Pierce Catalog2003-2004, page 306) using bovine serum albumin as a standard. Thehydrazide content was determined by TNBS assay using adipic aciddihydrazide as a standard, as described in Vidal, J. Immunol. Methods1986; 86:155-156. The degree of activation of TT so prepared wasapproximately 50 hydrazide groups per TT molecule.

Activation of Mn A PS to Contain Aldehyde Groups

Mn A PS (10 mg/mL in 25 mM HEPES, pH 7.4) was reacted with 6 mM NaIO₄ at20-24° C. for 4 hours. The sample was dialyzed against 10 mM HEPES, pH7.4 at 4° C. using a 12-14 KDa dialysis membrane. The concentration ofthe resulting activated PS was determined by phosphorus assay, asdescribed in Keleti et al, Handbook of micromethods for the biologicalsciences. 70. Phosphorus (Total). Page 84. Van Nostrand Reinhold Co.,New York, 1974. The aldehyde content of the activated PS was determinedby BCA assay (Pierce Catalog 2003-2004, pages 241 and 305) using glucoseas a standard. The degree of activation of the activated Mn A PSprepared by this protocol was approximately one aldehyde group per 80 to110 monomeric repeating units.

Conjugation of Activated Mn A PS to Activated TT

An aliquot of hydrazide-containing TT was adjusted to 10 mg/mL bylyophilization and dissolution in water. An aliquot ofaldehyde-containing Mn C PS was adjusted to 10 mg/mL by lyophilizationand dissolution in 0.2 M HEPES, pH 7.5, 30 mM EDTA. Activated TTsolution was added to equal volume of the activated Mn A PS andvortexed. The reaction mixture was incubated at 20-24° C. for 18 hours.The reaction mixture was treated with NaBH₄. (10-fold molar equivalentto initial aldehyde concentration in the activated PS) for 6 hours. Thesolution was buffer-exchanged with saline, 10 mM HEPES, pH 7.5, 1 mMEDTA using a 12-14 KDa molecular weight cut-off membrane. Total proteinwas determined by Lowry assay (see Pierce Catalog 2003-2004, page 306)using bovine serum albumin as a standard. The total Mn A PS content wasdetermined by phosphorus assay, as described in Keleti et al., Handbookof micromethods for the biological sciences. 70. Phosphorus (Total).Page 84. Van Nostrand Reinhold Co., New York, 1974. Several preparationsof Mn A PS-TT conjugates were made. The HPSEC profile of one of theseconjugates and the activated TT are shown in FIG. 6.

Immunogenicity of Mn A PS-TT Conjugates

These Mn A PS-TT conjugate preparations and native Mn A PS (control)were separately used to immunize groups of 10 mice at 1 μgpolysaccharide/dose on days 0 and 14. The geometric means of the inducedantibody levels (units/mL) two weeks post 2^(nd) injection are 79units/mL for native PS control group and 11,000-47,000 units/mL for theconjugate groups, assuming 3200 units/mL for the reference serum (Table5). The conjugates induced 169-595 fold more anti-Mn A PS specificantibody in mice as compared to the native Mn A PS control.

TABLE 5 Mouse groups of different conjugate and Geometric mean Foldpolysaccharide anti-MAPS antibody increase over immunogens level inunits/mL (CI)^(a) control group Native Mn A PS 79 (21, 290)  — (control)MA031209R 14,861 (6542, 33757) 188 MA031209B 13,375 (7677, 23303) 169MA031212B 14,777 (5433, 40191) 187 MA031212J 13,385 (5150, 34789) 169MA031216B 15,052 (4910, 46149) 191 MA031216J 11,074 (4605, 26628) 140MA031219R 20,410 (9282, 44884) 258 MA031219B 13,813 (4645, 41071) 175MA031219J 26,826 (9172, 78463) 340 MA031221R 18,994 (8012, 45030) 240MA031221B  42,041 (25208, 73147) 532 MA031221J  46,981 (20238, 109062)595 ^(a)The geometric mean anti-Mn A PS antibody levels (compared to areference serum with anti-Mn A PS antibody level of 3200 units/mL) with1 SD confidence interval of mouse groups (10 mice per group) two weekspost 2^(nd) immunization with 1 μg/dose native Mn A PS or each of thefour Mn A PS-TT conjugates.

Method B Pneumococcal Type 6B Conjugate

Activation of TT to Contain Hydrazide Groups

Tetanus toxoid (4.2 mg/mL) was activated with 0.42 M hydrazine in thepresence of 20 mM EDC, 0.1 M MES, pH 6.5 at 20-24° C. After reaction for4 hours, the pH of the reaction mixture was raised to 7.5-10 with 1 NNaOH to stop the reaction. The reaction mixture was buffer-exchangedwith 30 mM NaCl, 3 mM Na₂CO₃, pH about 10.5 at 4° C. using a 12-14 KDadialysis membrane. The protein concentration of the resultingTT-hydrazide sample was determined by Lowry assay (Pierce Catalog2003-2004, page 306) using bovine serum albumin as a standard. Thehydrazide content was determined by TNBS assay using adipic aciddihydrazide as a standard, as described in Vidal, J. Immunol. Methods1986; 86:155-156. The degree of activation of TT so prepared isapproximately 50 hydrazide groups per TT molecule.

Activation of Pn 6B PS to Contain Cyanate Groups

Pn 6B polysaccharide (0.4 mL, 10 mg/mL) was activated with 38 μL CDAP(100 mg/mL in acetonitrile) for 2-2.5 minutes at 20-24° C. in thepresence of 38 μL 0.2 M triethylamine. The activated polysaccharide wasmixed with 5 mL ice-cold 0.2 M HEPES, pH 7.5, 30 mM EDTA, andimmediately used for conjugation.

Conjugation of Activated Pn 6B PS to Activated TT

The activated polysaccharide was added to 2 mg activated TT (ice-cold,0.5 mL, 4 mg/mL); vortex. After incubating at 4° C. with gentle shakingfor 36 hours, the reaction mixture was incubated at 20-24° C. foranother 18 hours. The prolonged incubation ensured decomposition of anyresidual unreacted cyanate groups. The solution was buffer-exchangedwith saline, 10 mM HEPES, pH 7.5, 1 mM EDTA using a 12-14 KDa molecularweight cut-off membrane. The total protein content is determined byLowry assay (Pierce Catalog 2003-2004, page 306), and the totalpolysaccharide content was determined by anthrone assay, as described byKeleti et al., Handbook of micromethods for the biological sciences. 61.Hexoses (Anthrone). Page 73. Van Nostrand Reinhold Co., New York, 1974.The HPSEC profiles of Pn 6B PS-TT and the activated T are shown in FIG.7.

Immunogenicity of Pn 6B PS-TT Conjugate

The Pn 6B PS-TT conjugate as prepared above and native Pn 6B PS(control) were separately used to immunize groups of 10 mice at 1 μgpolysaccharide/dose on days 0, 14 and 28. The geometric means of theinduced antibody levels (units/mL) two weeks post 3^(rd) injection were13 units/mL for native Pn 6B PS control group and 3,700 units/mL for thePn 6B PS-TT conjugate group, assuming 3200 units/mL for the referenceserum (Table 6). The conjugate induced 285 fold anti-Pn 6B PS specificantibody in mice as compared to the native Pn 6B PS control.

TABLE 6 Mouse groups of different Geometric conjugate and mean anti-MAPSFold polysaccharide antibody level increase over immunogens in units/mL(CI)^(a) control group Native Pn 6B PS 13 (10, 17)  — (control) Pn 6BPS-TT 3,700 (240, 5,705) 285 ^(a)The geometric mean anti-Pn 6B PSantibody levels (compared to a reference serum with anti-Pn 6B PSantibody level of 3200 units/mL) with 1 SD confidence interval of mousegroups (10 mice per group) two weeks post third immunization with 1μg/dose native Pn 6B PS or the Pn 6B PS-TT conjugate.

Method B Pneumococcal Type 7F Conjugate

Activation of TT to Contain Hydrazide Groups

Tetanus toxoid (4.2 mg/mL) was activated with 0.42 M hydrazine in thepresence of 20 mM EDC, 0.1 M MES, pH 6.5 at 20-24° C. After reaction for4 hours, the pH of the reaction mixture was raised to 7.5-10 with 1 NNaOH to stop the reaction. The reaction mixture was buffer-exchangedwith 30 mM NaCl, 3 mM Na₂CO₃, pH about 10.5 at 4° C. using a 12-14 KDadialysis membrane. The protein concentration of the resultingTT-hydrazide sample was determined by Lowry assay (Pierce Catalog2003-2004, page 306) using bovine serum albumin as a standard. Thehydrazide content was determined by TNBS assay using adipic aciddihydrazide as a standard, as described in Vidal, J. Immunol. Methods1986; 86:155-156. The degree of activation of TT so prepared wasapproximately 50 hydrazide groups per TT molecule.

Activation of Pn 7F PS to Contain Cyanate Groups

Pn 7F polysaccharide (0.4 mL, 10 mg/mL) was activated with 38 μL CDAP(100 mg/mL in acetonitrile) for 2-2.5 minutes at 20-24° C. in thepresence of 38 μL 0.2 M triethylamine. The activated polysaccharide wasmixed with 5 mL ice-cold 0.2 M HEPES, pH 7.5, 30 mM EDTA, andimmediately used for conjugation.

Conjugation of Activated Pn 7F PS to Activated TT

The activated polysaccharide was added to 2 mg activated TT (ice-cold,0.5 mL, 4 mg/mL) and vortexed. After incubating at 4° C. with gentleshaking for 36 hours, the reaction mixture was incubated at 20-24° C.for another 18 hours. The prolonged incubation ensured decomposition ofany residual unreacted cyanate groups. The solution was buffer-exchangedwith saline, 10 mM HEPES, pH 7.5, 1 mM EDTA using a 12-14 KDa molecularweight cut-off membrane. Total protein content was determined by Lowryassay (Pierce Catalog 2003-2004, page 306). Total polysaccharide contentwas determined by anthrone assay, as described by Keleti et al.,Handbook of micromethods for the biological sciences. 61. Hexoses(Anthrone), Page 73, Van Nostrand Reinhold Co., New York, 1974. TheHPSEC profiles of Pn 7F PS-TT and the activated TT are shown in FIG. 8.

Immunogenicity of Pn 7F PS-TT Conjugate

The Pn 7F PS-TT conjugate prepared as described above and native Pn 7FPS (control) were separately used to immunize groups of 10 mice at 1 μgpolysaccharide/dose on days 0, 14 and 28. The geometric means of theinduced antibody levels (units/mL) two weeks post 3^(rd) injection are17 units/mL for native Pn 7F PS control group and 17,077 units/mL forthe Pn 7F PS-TT conjugate group, assuming 3200 units/mL for thereference serum (Table 7). The conjugate induced 1,005 fold anti-Pn 7FPS specific antibody in mice as compared to the native Pn 7F PS control.

TABLE 7 Mouse groups of different Geometric conjugate and mean anti-MAPSFold polysaccharide antibody level increase over immunogens in units/mL(CI)^(a) control group Native Pn 7F PS 17 (14, 20)   — (control) Pn 7FPS-TT 17,077 (8,034, 36,299) 1,005 ^(a)The geometric mean anti-Pn 7F PSantibody levels (compared to a reference serum with anti-Pn 7F PSantibody level of 3200 units/mL) with 1 SD confidence interval of mousegroups (10 mice per group) two weeks post third immunization with 1μg/dose native Pn 7F PS or the Pn 7F PS-TT conjugate.

Method C Pneumococcal Serotype 9V Conjugate

Activation of TT to Contain Aldehyde Groups

Tetanus toxoid (4.2 mg/mL) was activated with 0.42 M1-amino-2,3-propanediol (APDO) in the presence of 20 mM EDC, 0.1 M MES,pH 6.5 at 20-24° C. After reacting for 4 hours, the pH of the reactionmixture was raised to 7.5-10 with 1 N NaOH to stop the reaction. Thereaction mixture was buffer-exchanged with 30 mM NaCl, 3 mM Na₂CO₃, pHabout 10.5 at 4° C. using a 12-14 KDa dialysis membrane. The degree ofTT modification with APDO is determined by purpald assay (as describedin Lee et al., Anal. Biochem. 2001; 296:73-82) and Lowry assay (PierceCatalog 2003-2004, page 306). An aliquot of TT-APDO was reacted with 6mM NaIO₄ for 1 hour and then buffer exchanged with 30 mM NaCl, 3 mMNa₂CO₃, pH about 10.5. The degree of activation of TT prepared wasapproximately 26 APDO or aldehyde groups per TT molecule.

Activation of Pn 9V PS to Contain Hydrazide Groups

Pn 9V PS (0.4 mL, 10 mg/mL) was activated with 36 μL CDAP (100 mg/mL inacetonitrile) for 2-2.5 minutes at 20-24° C. in the presence of 36 μL0.2 M triethylamine. At the end of activation, 0.4 mL 0.5 M ADH wasadded and mixed. The reaction mixture was incubated 18 hours at 20-24°C. The sample was dialyzed against 10 mM HEPES, pH 7.5 at 4° C. using a12-14 KDa dialysis membrane. The PS concentration is determined byanthrone assay, as described in Keleti et al., Handbook of micromethodsfor the biological sciences. 61. Hexoses (Anthrone), Page 73. VanNostrand Reinhold Co., New York, 1974. The hydrazide content wasdetermined by TNBS assay using adipic acid dihydrazide as a standard, asdescribed in Vidal, J. Immunol. Methods 1986; 86:155-156. The degree ofactivation of the activated Pn 9V PS prepared by this protocol wasapproximately one hydrazide group per saccharide repeating unit.

Conjugation of Activated Pn 9V PS to Activated TT

An aliquot of hydrazide-containing Pn 9V PS (1 mg; 0.236 mL 4.233 mg/mL)was mixed with 0.067 mL 1 M HEPES, pH 7.5 and 0.068 mL H₂O. An aliquotof aldehyde-containing TT (1 mg; 0.296 mL 3.38 mg/mL) was added to theactivated Pn 9V PS (Total volume, 0.67 mL; initial concentration forboth reactants, 1.5 mg/mL). The reaction mixture was incubated at 20-24°C. for 18 hours. The reaction mixture was treated with NaBH₄ (10-foldmolar equivalent to initial aldehyde concentration in the activated PS)for 6 hours. The solution was buffer-exchanged with saline, 10 mM HEPES,pH 7.5, 1 mM EDTA using a 12-14 KDa molecular weight cut-off membrane.Total protein was determined by Lowry assay (Pierce Catalog 2003-2004,page 306) using bovine serum albumin as a standard. Total Pn 9V PScontent was determined by anthrone assay, as described by Keleti et al.,Handbook of micromethods for the biological sciences. 61. Hexoses(Anthrone), Page 73, Van Nostrand Reinhold Co., New York, 1974. TheHPSEC profiles of Pn 9V PS-TT and the activated TT are shown in FIG. 9.

Immunogenicity of Pn 9V PS-TT Conjugate

These Pn 9V PS-TT conjugate and native Pn 9V PS (control) wereseparately used to immunize groups of 10 mice at 1 μgpolysaccharide/dose on days 0, 14 and 28. The geometric means of theinduced antibody levels (units/mL) two weeks post 3^(rd) injection are15 units/mL for native Pn 9V PS control group and 13,291 units/mL forthe Pn 9V PS-TT conjugate group, assuming 3200 units/mL for thereference serum (Table 8). The conjugate induced 886 fold anti-Pn 9V PSspecific antibody in mice as compared to the native Pn 9V PS control.

TABLE 8 Mouse groups of different Geometric conjugate and mean anti-MAPSFold polysaccharide antibody level increase over immunogens in units/mL(CI)^(a) control group Native Pn 9V PS 15 (14, 16)   — (control) Pn 9VPS-TT 13,291 (6,339, 27,869) 886 ^(a)The geometric mean anti-Pn 9V PSantibody levels (compared to a reference serum with anti-Pn 9V PSantibody level of 3200 units/mL) with 1 SD confidence interval of mousegroups (10 mice per group) two weeks post third immunization with 1μg/dose native Pn 9V PS or the Pn 9V PS-TT conjugate.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention as embodied in the attached claims.

What is claimed is:
 1. A method for preparing a conjugate vaccine, themethod comprising: reacting a polysaccharide with an oxidizing agent,whereby a solution of an aldehyde-activated polysaccharide is obtained;buffer exchanging the solution of the aldehyde-activated polysaccharideto a pH of from 7 to 8; reacting a protein with carbohydrazide or adihydrazide in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride at a pH of from 6 to 7, whereby a solution ofan hydrazide-activated protein is obtained; raising a pH of the solutionof the hydrazide-activated protein to from 7.0 to 11; buffer exchangingthe solution of the hydrazide-activated protein to a pH of from 10.0 to11.0; reacting the aldehyde-activated polysaccharide with thehydrazide-activated protein at a pH of from 6 to 8, whereby a conjugatecomprising one or more C═N double bonds is obtained; and reducing theC═N double bonds of the conjugate to C—N single bonds, whereby aconjugate vaccine capable of stimulating an immune response is obtained.2. The method according to claim 1, wherein the oxidizing agentcomprises NaIO₄.
 3. The method according to claim 1, wherein thesolution of the aldehyde-activated polysaccharide is buffer exchangedwith a HEPES buffer.
 4. The method according to claim 1, wherein thealdehyde-activated polysaccharide is reacted with thehydrazide-activated protein at a ratio of from about 1:2 to about 2:1.5. The method according to claim 1, wherein reducing comprises reducingwith NaBH₄.
 6. The method according to claim 1, wherein thepolysaccharide is selected from the group consisting of Meningococcalpolysaccharides, Pneumococcus polysaccharides, Hemophilus influenzaetype b polysaccharide, Vi polysaccharide of Salmonnella typhi, and groupB Streptococcus polysaccharides.
 7. The method according to claim 1,wherein the protein is selected from the group consisting of tetanustoxoid, diphtheria toxoid, CRM₁₉₇, and meningococcal protein.
 8. Themethod according to claim 1, wherein the solution of thehydrazide-activated protein is buffer-exchanged at a pH of from 10.3 to10.7.
 9. The method according to claim 1, wherein the solution of thehydrazide-activated protein is buffer-exchanged with a3-(cyclohexylamino)-1-propanesulfonicacid buffer or a(2-(N-cyclohexylamino)ethane sulfonic acid buffer.
 10. The methodaccording to claim 1, wherein the dihydrazide is succinyl dihydrazide.